| Part Ⅰ Rapid and simultaneous determination of ten anti-tuberculosis drugs in human plasma by UPLC-MS/MS with applications in drug monitoringObjective: To establish a novel sensitive high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for simultaneous detection of ten anti-tuberculosis drugs in human plasma including isoniazid(INH),rifampicin(RIF),ethambutol(EMB),pyrazinamide(PZA),rifampin(RFB),rifapentine(RFP)as well as four second-line antituberculosis drugs,protionamide(PTO),ethionamide(ETH),thiosemicarbazone(THS),and clofazimine(CFZ),and to apply it successfully to monitoring of the concentration of 10 target drugs in the blood of pulmonary tuberculosis(TB)patients.Method: The plasma samples were treated with acetonitrile to precipitate proteins,and doped with the isotope internal standard.A Shiseido CAPCELL RAK-ADME(2.1 mm × 50 mm,3 μm)column was used for chromatographic separation,acetonitrile-water(containing 0.1% formic acid)as the mobile phase.The separation used gradient elution with a flow rate of 0.4 m L/min.The column temperature was 40 °C,and the sample volume was 1.0 μL.The electrospray ionization source(ESI)and the positive ion multiple reaction monitoring(MRM)mode were used for detection.The analysis time was as short as 7 min.Results: Under the optimal conditions,the limit of detections(LODs)and the limit of quantitation(LOQs)of ten anti-tuberculosis drugs were in the range of 0.03～3.33 ng/m L and 0.10～10.00 ng/m L,respectively.The spiked recovery was within the range 91.21%～122.10,the intraday precision ≤ 6.52 and the interday precision ≤ 11.58.indicating the method good accuracy and precision.Conclusion: This study offers important advances for the simultaneous detection of ten kinds of anti-TB drugs within 7 min reported.It is applicable for therapeutic drug monitoring,and the result is satisfactory.Part Ⅱ Detection of caffeine and its main metabolites in plasma using high performance liquid chromatography tandem mass spectrometry for early diagnosis of Parkinson’s diseaseObjective: To establish a novel,sensitive and selective HPLC-MS/MS method for determining of the levels of caffeine(CA)and its three main downstream metabolites–paraxanthine(PX),theobromine(TB)and theophylline(TP)in human plasma,and successfully apply it to the early auxiliary diagnosis of Parkinson’s disease(PD).Method: An ACQUITY UPLC HSS T3(2.1 mm×100 mm,1.8 μm)column was used for chromatographic separation,and methyl alcohol-water as the mobile phase.The separation used gradient elution with a flow rate of 0.4m L/min.The column temperature was 40 °C,and the sample volume was 1.0μL.The electrospray ionization source(ESI)and the positive ion multiple reaction monitoring(MRM)mode were used for the detection.The serum samples were purified and enriched by Chem Elut S solid supported liquid-liquid extraction column and quantified by isotope internal standard method.Results: The results showed that under the optimized conditions,there was a good linear relationship in the range of 0.5～1000.0 ng/m L for CA,2.0～750.0 ng/m L for TB,0.5～750.0 ng/m L for PX and TP,with the linear correlation coefficient of r > 0.9996,and the LODs of the four targets were0.12 ng/m L for CA,1.25 ng/m L for TB,0.12 ng/m L for PX and 0.12 ng/m L for TP,respectively.The recovery was within the range of 83.2%～97.9%,the intraday precision ≤ 6.52% and the interday precision ≤ 11.58%(n=5).Conclusion: A novel,sensitive and selective method was developed to determine the CA and its metabolites in plasma,and the result is satisfactory,suggesting that the present method could serve as a practical early diagnostic in PD patients.Part Ⅲ A metabolomics approach based on gas-mass spectrometry for the discovery of biomarkers for early diagnosis of Parkinson’s diseaseObjective: To establish a new approach combining untargeted and targeted metabolomics used for determining reliable and stable differential markers for early clinical diagnosis of Parkinson’s disease.Method: This study consists of two stages: discovery and verification.Firstly,80 samples from discovery cohort were analyzed by gas chromatography-mass spectrometry(SPME-GC-TOFMS)combining solid-phase microextraction and 304 substance were identified out,then a multivariate statistical analysis of all identified substances was performed.When the nonparametric test Wilcoxon-Mann-Whitney test P < 0.5 and PLS-DA model VIP > 1,differences metabolites were filter out,then the ROC curve analysis was carried out and 6 biomarkers were successfully screened.Finally,the samples in the validation cohort were targeted and quantitatively detected by purge and trap(P&T)-GCMS to verify the biomarkers selected in the discovery cohort.Results: In this study,non-targeted metabonomics method based on SPME-GC-TOFMS was used to screen out 6 differential biomarkers,in which a-curcumene,hexadecanol,Vanillin and dodecane were upregulated while o-chlorobenzaldehyde and 2,6-ditert-butyl phenol were downregulated.The targeted metabonomics method of P&T-GCMS was used to verify the six differential biomarkers screened from the discovery cohort,and three biomarkers and the differential metabolite indole were selected for targeted verification.The results were consistent with the downward trend in the discovery group.Conclusion: In this study,the combination approach of untargeted and targeted metabolomics was used to effectively identify different biomarkers,which provided reliable technical support for clinical diagnosis of early Parkinson’s disease. |
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